NMI-1182, a gastro-protective cyclo-oxygenase-inhibiting nitric oxide donor

Inflammopharmacology, Vol. 12, No. 5–6, pp. 521– 534 (2005)  VSP 2005.

Also available online - www.vsppub.com

NMI-1182, a gastro-protective cyclo-oxygenase-inhibiting nitric oxide donor JAMES L. ELLIS ∗ , MICHAEL E. AUGUSTYNIAK, EDWARD D. COCHRAN, RICHARD A. EARL, DAVID S. GARVEY, LAURA J. GORDON, DAVID R. JANERO, SUBHASH P. KHANAPURE, L. GORDON LETTS, TERRY L. MELIM, MADHAVI G. MURTY, DAVID J. SCHWALB, MATTHEW J. SHUMWAY, WILLIAM M. SELIG, A. MARK TROCHA, DELANO V. YOUNG and IRINA S. ZEMTSEVA NitroMed Inc., 125 Spring Street, Lexington, MA 02421-0781, USA Accepted 28 January 2005 Abstract—Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to treat inflammation and to provide pain relief but suffer from a major liability concerning their propensity to cause gastric damage. As nitric oxide (NO) is known to be gastro-protective we have synthesized a NO-donating prodrug of naproxen named NMI-1182. We evaluated two cyclo-oxygenase (COX)inhibiting nitric oxide donors (CINODs), NMI-1182 and AZD3582, for their ability to be gastroprotective compared to naproxen and for their anti-inflammatory activity. NMI-1182 and AZD3582 were found to produce similar inhibition of COX activity to that produced by naproxen. Both NMI1182 and AZD3582 produced significantly less gastric lesions after oral administration than naproxen. All three compounds effectively inhibited paw swelling in the rat carrageenan paw edema model. In the carrageenan air pouch model all three compounds significantly reduced PGE2 levels in the pouch exudate but only NMI-1182 and naproxen inhibited leukocyte influx. These data demonstrate that NMI-1182 has comparable anti-inflammatory activity to naproxen but with a much reduced likelihood to cause gastric damage. Key words: Non-steroidal anti-inflammatory drug (NSAID); nitric oxide; gastro-protection; gastric damage; naproxen; CINOD; anti-inflammatory; cyclo-oxygenase.

1. INTRODUCTION

Non-steroidal anti-inflammatory drugs (NSAIDs) remain a mainstay in the treatment of inflammation, pyresis and pain. Despite their widespread use in these ther∗ To

whom correspondence should be addressed. Tel.: (1-761) 266-4132; Fax: (1-761) 274-8083; e-mail: [email protected]

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apeutic areas, their long-term administration is limited because of their propensity to produce adverse gastrointestinal effects including dyspepsia, gastric erosions, ulceration and bleeding (James and Hawkey, 2003). Due to their common use and their propensity to produce ulceration and bleeding it has been estimated that NSAIDs contribute to as many as 16 000 deaths per year in the USA (Wolfe et al., 1999) and 2000 per year in the UK (Tramer et al., 2000). NSAIDs exert their pharmacological action by reducing the production of prostaglandin synthesis through inhibition of the enzyme cyclo-oxygenase (COX). The discovery that COX exists in at least two distinct isoforms, termed COX-1 and COX-2, and that typical NSAIDs do not discriminate between the two isoforms led to considerable studies on the role of the two isoforms in the regulation of prostaglandin synthesis and biology under normal and pathological conditions. The hypothesis that NSAIDs’ therapeutic benefit comes from their ability to inhibit the inducible COX-2 isoform while their adverse gastrointestinal (GI) effects come from inhibition of the constitutive COX-1 isoform (Vane et al., 1998; Mitchell and Warner, 1999) has led to an extensive drug discovery effort to identify COX-2selective inhibitors (Flower, 2003). As a result of this effort several COX-2-selective inhibitors (coxibs), typified by rofecoxib, celecoxib and valdecoxib, have been synthesized and characterized and marketed in recent years (de Leval et al., 2004). Coxibs have been found to be efficacious anti-inflammatory agents exhibiting a reduced incidence of GI adverse events when compared to NSAIDs (Bombardier et al., 2000; Silverstein et al., 2000; Lisse et al., 2003). This reduced GI event profile, however, is lost in patients who take concomitant aspirin, even at low dose (Silverstein et al., 2000; Fiorucci et al., 2003; Laine et al., 2004). Selective inhibition of COX-2 may also have led to some unexpected cardiovascular events. There is a growing body of evidence that selective COX-2 inhibitors may increase the risk of having a severe cardiovascular event (Bombardier et al., 2000; Reicin et al., 2002). This has led to the recent withdrawal of Vioxx
from the market and a review of coxib safety by the FDA (2005). There therefore remains a need to develop drugs which have the therapeutic efficacy of NSAIDs but are devoid of gastrointestinal and cardiovascular liabilities. One possible solution to the aforementioned pathophysiology associated with chronic NSAID use may be the addition of a nitric oxide (NO) donating moiety to existing NSAIDs. NO has been shown to maintain the integrity of GI epithelium under pathophysiological conditions such as NSAID and coxib toxicity (Muscara et al., 1998; Ranatunge et al., 2004). We have synthesized NO-donating prodrugs of naproxen. Naproxen is known to be an efficacious NSAID and evidence suggests that it may confer cardio-protective benefits versus the coxibs and other NSAIDs (Bombardier et al., 2000). In the present study, we have compared the in vitro and in vivo activity of NMI-1182, a prototypic dinitrate NO-naproxen, with the mononitrate NO-naproxen (AZD3582) and naproxen itself (see Fig. 1 for structures) in well-established rat carageenan

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Figure 1. Chemical structures of NMI-1182, AZD3582, Naproxen Na and des-NO-NMI-1182.

inflammation models and also for their ability to produce gastric lesions following acute dosing in the rat.

2. MATERIALS AND METHODS

2.1. Animals and materials Male Sprague–Dawley rats (180–220 g) were obtained from Charles River (Kingston, NY, USA). Animals were barrier housed in a clean environment with access

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to water and food ad libitum, and kept in a temperature controlled environment (20–22◦ C and 40–60% relative humidity) on a 12 : 12 light/dark cycle. All procedures used were approved by NitroMed’s Institutional Animal Care and Use Committee. Naproxen-Na was obtained from Sigma (St. Louis, MO, USA). NMI1182, AZD3582 and des-NO-NMI-1182 were synthesized by the Chemistry group at NitroMed. 2.2. Cycloooxygenase inhibition: human whole blood assay Fresh heparinized human blood was obtained by informed consent from non-fasted, male or female donors who had not taken any aspirin, NSAIDs or coxibs for 14 days. Blood was distributed in 1-ml aliquots per well of a 24-well tissue culture plate. The plate was placed on a gently rotating platform shaker in a 5% CO2 incubator at 37◦ C for 15 min. The test compounds (naproxen-Na, NMI-1182, des-NO-NMI1182 and AZD3582) were dissolved and diluted in DMSO and 1 µl of each dilution of test compound or corresponding vehicle was added per well to duplicate wells. To induce COX-2, lipopolysaccharide (LPS) from Escherichia coli (Sigma) was added at 10 µg/ml to appropriate wells 15 min after addition of test compounds. To stimulate COX-1, the calcium ionophore A23187 (Sigma) was added (25 µM) to separate wells 285 min after the addition of test compounds. 30 min after the A23187 addition or 300 min after LPS addition, all incubations were terminated by cooling on ice and addition of 2 mM EGTA. The blood samples were centrifuged at 1200 × g for 10 min at 4◦ C. Following centrifugation, 100 µl of the plasma was added to 1 ml of methanol, mixed vigorously and stored overnight at −20◦ C. The following day, samples were centrifuged at 200 × g for 10 min at 4◦ C. The resulting supernatants were evaporated to dryness. After reconstitution with EIA buffer and appropriate dilution (2000-fold for COX-1 and 500-fold for COX-2) samples were assayed in duplicate for thromboxane B2 (TXB2 ) using EIA kits supplied by Cayman (Ann Arbor, MI, USA). Results are expressed as the mean ± SEM. 2.3. Rat aorta Male Sprague–Dawley rats (180–220 g) were anesthetized with ketamine (10 mg/kg, i.p.), exsanguinated and their abdominal aorta were removed. After the removal of fat and connective tissue, the aorta was cut in rings, 3–4 mm in length, and suspended in 10 ml organ baths containing a modified Krebs’ solution (composition in mM: NaCl 119, CaCl2 2.5, KCl 4.5, NaHCO3 25, MgCl2 1.25, NaHPO4 1.0, D -glucose 11.1). The Krebs’ solution was maintained at 37◦ C and gassed with 5% CO2 in O2 . The aortic rings were suspended at an initial resting tension of 1.5 g and washed every 15 min during a 60-min equilibration period. After the equilibration period, the rings were contracted with 1 µM phenylephrine (Sigma) and the contraction was allowed to reach a plateau before test compounds dissolved in DMSO were administered in a cumulative fashion (1 nM–10 µM). Results were calculated as a percentage of the maximum relaxation produced by 10 µM s-nitrosoglutathione (GSNO) and are expressed as the mean ± SEM.

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2.4. Plasma naproxen levels Male Sprague–Dawley rats (180–220 g) were fasted overnight with access to water ad libitum. The test compounds, NMI-1182, AZD3582 and naproxen-Na (1– 10 mg/kg naproxen-Na molar equivalents), were suspended in 0.5% methocel (Dow Chemical, Midland, MI, USA) immediately prior to oral gavage. The rats were killed 5 h post-dosing and blood was collected by cardiac puncture of the left ventricle into a 3-ml heparinized syringe and then placed into a labeled microfuge tube. Plasma was prepared by centrifugation at 2000 × g for 15 min at 4◦ C followed by extraction 1 : 1 with acetonitrile on ice. Samples were then vortexed three times over a 15 min period followed by centrifugation to remove any particulate matter at 16 000 × g for 20 min at 4◦ C. The supernatant was filtered using a 0.25 µm PTFE filter for subsequent HPLC analysis on a Beckman 266 pump module (Beckman Coulter, Fullerton, CA, USA) with a Beckman 166 UV detector (Beckman Coulter). The analysis column was a Waters Bondapak C18 (3.9 × 300 mm, 10 µm particle size; Waters, Milford, MA, USA) at ambient temperature. The mobile phase for column separation was 60% acetonitrile, 40% water, pH 3.3 adjusted with glacial acetic acid. Flow rate was 2 ml/min and injection volume was 100 µl. Results are expressed as the mean ± SEM. 2.5. Gastric injury model Male Sprague–Dawley rats (180–220 g) were fasted overnight with access to water ad libitum. The test compounds, NMI-1182, des-NO-NMI-1182, AZD3582 and naproxen-Na (1–40 mg/kg naproxen-Na molar equivalents), were dissolved in PEG 400 (Sigma) immediately prior to oral gavage. Rats were killed 3 h postdosing. Their stomachs were removed, opened along the greater curvature and digitally photographed. The images were analyzed for visible hemorrhagic lesions by an investigator blinded to treatment, using Image-J software (NIH, Bethesda, MD, USA). The total length of all lesions for each stomach was summed and reported as the total lesion score. Data are expressed as means ± SEM. Statistical analysis was evaluated using a one-way analysis of variance (ANOVA), followed by Dunnett’s Multiple Comparison Test. P < 0.05 was considered significant. 2.6. Carrageenan air pouch model A pouch was formed in the intrascapular region of male Sprague–Dawley rats (180–220 g) by injection of sterile air on days −6 and −3 as previously described (Massferrer et al., 1994). On day 0, test compounds, NMI-1182, AZD3582, naproxen-Na or vehicle (0.5% methocel; Dow Chemical), was administered orally at 5 mg/kg (naproxen-Na molar equivalents), 1 h prior to the carrageenan injection (1.0 ml of a 1% solution) into the pouch. After 4 h, the animals were killed with CO2 and the inflammatory exudates were collected from the pouch for determination of prostaglandin E2 (PGE2 ) levels by immunoassay (Cayman). The number of

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leukocytes in the exudates was determined by a Beckman Coulter Z1 Particle Counter (Beckman Coulter). All data are expressed as means ± SEM. Statistical analysis was evaluated using a one-way analysis of variance (ANOVA), followed by Dunnett’s Multiple Comparison Test. P < 0.05 was considered significant. 2.7. Carrageenan paw inflammation Male Sprague–Dawley rats (180–220 g) were fasted overnight with access to water ad libitum. Test compounds, NMI-1182, des-NO-NMI-1182, AZD3582 and naproxen-Na (10 mg/kg naproxen-Na molar equivalents), were suspended in 0.5% methocel. The test compounds were administered by oral gavage 1 h prior to the carrageenan (Sigma) injection (50 µl 1% carrageenan in saline) into the sub plantar region of the right hind-paw pad. Paw volume was determined using the water displacement method (Winter et al., 1962) as the average of two measurements differing by less than 0.2 ml. Paw volume measurements were assessed immediately prior to and 3 h post carrageenan injection. Results are calculated as the % inhibition of the change in paw volume produced by the carrageenan injection and are expressed as means ± SEM. 3. RESULTS

3.1. Inhibition of human COX-1 and COX-2 NMI-1182, AZD3582 and naproxen-Na all showed similar efficacy and potency in their ability to inhibit both COX-1 and COX-2 activity, as measured in the human whole blood assay (Fig. 2). The − log IC50 for NMI-1182 was 4.71 ± 0.53 and 5.05 ± 0.30 for COX-1 and COX-2 inhibition, respectively. The − log IC50 for AZD3582 was 4.74 ± 0.35 and 5.09 ± 0.35 for COX-1 and COX-2 inhibition, respectively. The − log IC50 for naproxen-Na was 4.24 ± 0.61 and 4.97 ± 0.32 for COX-1 and COX-2 inhibition, respectively. 3.2. In vitro relaxant effect in rat aorta The ability of NMI-1182 and AZD3582 to relax rat isolated aorta (endothelium intact) pre-contracted with phenylephrine (1 µM) was compared. Results are expressed relative to maximal relaxation elicited by 10 µM GSNO. As illustrated in Fig. 3, NMI-1182 produced a concentration-dependent vasorelaxation that was greater than that produced by AZD3582. The vasorelaxation produced by AZD3582 was no different from vehicle (DMSO) control. Naproxen-Na or des-NO-NMI-1182 did not produce any relaxations at concentrations up to 30 µM (data not shown). 3.3. Plasma levels of naproxen following administration of NMI-1182, AZD3582 and naproxen-Na Plasma levels of naproxen were readily detectable at 5 h following oral administration of naproxen-Na (1–10 mg/kg), as well as molar equivalent doses of orally ad-

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Figure 2. Inhibition of (A) COX-1- and (B) COX-2 TXB2 production in human whole blood by NMI-1182 (1), AZD3582 (!) and naproxen ("). The data is expressed as % inhibition and is shown as the mean ± SEM, n = 6.

ministered NMI-1182 and AZD3582 (Fig. 4). Plasma levels of naproxen following administration of all three drugs showed dose-dependent increases with a tendency for the levels following NMI-1182 and AZD3582 administration to be lower than those produced after administration of naproxen-Na (Fig. 4). 3.4. Gastric lesion formation in the rat Fasted male Sprague–Dawley rats were orally treated with NMI-1182, AZD3582, naproxen-Na or the des-NO-NMI-1182 compound (5–40 mg/kg naproxen-Na molar equivalent in PEG 400). At 3 h following administration, animals were killed and their stomachs were scored for lesions. As shown in Fig. 5, naproxen-Na produced a dose-dependent increase in gastric lesions. In contrast, there was a significant reduction in gastric lesion formation in both NMI-1182- and AZD3582-treated animals when compared to the naproxen-Na-treated group. des-NO-NMI-1182 produced lesions similar in magnitude to those produced by naproxen-Na (Fig. 5). 3.5. Carrageenan-induced paw oedema in the rat Fasted male Sprague–Dawley rats were orally treated with NMI-1182, AZD3582 or naproxen-Na (at 10 mg/kg naproxen-Na molar equivalent in 0.5% methocel) 1 h prior to subplantar injection of carrageenan (50 µl of a 1% solution). Paw volumes

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Figure 3. Relaxant activity of NMI-1182 (1), AZD3582 (!), vehicle (DMSO, ") and GSNO (2) in the rat isolated aorta. Aorta were contracted with phenylephrine (1 µM). Results are calculated as the percent relaxation of the maximum relaxation produced by 10 µM GSNO and are given as the mean ± SEM, n = 6.

Figure 4. Naproxen levels in rat plasma 5 h following oral dosing of equimoral doses (1–10 mg/kg of naproxen NA) of naproxen ("), NMI-1182 (1) and AZD3582 (!). Results are shown as the mean ± SEM, n = 7–13.

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Figure 5. Gastric lesions scores in the rat following oral administration of equimolar doses (5– 40 mg/kg naproxen Na) of naproxen ("), NMI-1182 (1), AZD3582 (!) and des-NO-NMI-1182 (2). Results are shown as the mean ± SEM, n = 7–13. ∗∗ P < 0.01, ∗∗∗ P < 0.001 compared to naproxen.

Figure 6. Inhibition of carageenan-induced paw edema in the rat following oral administration of equimolar doses (10 mg/kg naproxen Na) of naproxen ("), NMI-1182 (1), AZD3582 (!). Results are shown as the mean ± SEM, n = 10–13.

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Figure 7. Effect of oral administration of equimoral doses (5 mg/kg naproxen Na) of naproxen, NMI1182 and AZD2382 on the inflammatory response in the rat carageenan air pouch model. Results are shown for PGE2 levels (open bars) and for leukocyte counts (solid bars) in the pouch exudates and are given as the mean ± SEM, n = 7–9. ∗∗ P < 0.01 compared to vehicle.

were assessed 1–6 h post-dosing. As shown in Fig. 6, all the compounds tested showed a comparable inhibition of between 50 and 65% at 3 h post-insult. 3.6. Carrageenan-induced air pouch inflammation Fasted male Sprague–Dawley rats, in which a 6-day air pouch was formed, were orally treated with NMI-1182, AZD 3582 or naproxen-Na (at 1–10 mg/kg naproxenNa equivalent in 0.5% methocel) 1 h prior to intra-pouch injection of carrageenan (1 ml of a 1% solution). At 4 h post-insult, pouch exudates were collected to assess inflammation (leukocyte count) and to measure PGE2 levels. As shown in Fig. 7, NMI-1182 significantly reduced carrageenan-induced leukocyte influx, whereas AZD3582 did not. Naproxen-Na, NMI-1182 and AZD3582 all significantly reduced PGE2 levels in the exudates by 93, 90 and 70%, respectively (Fig. 7).

4. DISCUSSION

Ever since the NSAIDs were introduced there has been considerable effort to develop agents which retain their desirable anti-inflammatory, analgesic and antipyretic properties but which have an improved gastric tolerability. Recently, two

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strategies have been evaluated with some success. The first was the development of agents which were selective for inhibiting the COX-2 isoform. This strategy was based on the hypothesis that the inducible COX-2 isoform was responsible for prostanoid production during inflammation whereas the constitutive COX-1 isoform produced “housekeeping” prostanoids, such as PGE2 , which are involved in mucosal integrity in the GI tract (Vane et al., 1998; Mitchell and Warner 1999). The first three COX-2-selective agents or coxibs (celecoxib, rofecoxib and valdecoxib) gained rapid success and met their goal in providing equal therapeutic benefit to the NSAIDs along with an improved GI profile (Bombardier et al., 2000; Silverstein et al., 2000; Brune and Hinz, 2004). Recent evidence suggests, however, that their improved GI profile is abolished when the coxibs are used in combination with aspirin, even at low dose (Silverstein et al., 2000; Fiorucci et al., 2003; Laine et al., 2004). One unexpected and controversial result of making compounds selective for COX2 over COX-1 is that the coxibs have been recently found to produce a higher incidence of serious adverse cardiovascular events (stroke and myocardial infarction) compared to NSAIDs and to placebo (Bombardier et al., 2000; Mukherjee et al., 2001; Reicin et al., 2002). Rofecoxib was found to produce a 2-fold increase in serious cardiovascular events compared to placebo in a recent trial looking at its potential benefit in colon polyp production, a finding which led Merck to withdraw rofecoxib from the market. Very recent evidence suggests that others in the coxib class, like celecoxib and valdecoxib, also carry an increased risk of precipitating adverse cardiovascular events (Ott et al., 2003; Couzin, 2004). The second strategy to produce effective anti-inflammatory agents with an improved side effect profile has been to add NO-donating functionality to NSAIDs, as NO should provide benefit through a multiple of mechanisms, including (1) increased gastric mucosal blood flow, (2) inhibition of leukocyte adhesion or activation (Kubes et al., 1991), (3) increased mucus secretion or gel thickness via a cGMP-dependent mechanism (Brown et al., 1992, 1993), (4) inhibition of gastric acid secretion and (5) scavenging of reactive oxygen species (Kwiecien et al., 2002). NO may, therefore, mimic the actions of beneficial or cytoprotective prostaglandins, thus allowing it to compensate for decreased formation of “housekeeping” prostanoids such as PGE1 or PGI2 . This allows NO to act as a physiological substitute for the prostaglandins that are lost, as occurs when their production is inhibited by NSAIDs. One compound from this class of NO-donating NSAIDs, AZD3582, has entered clinical trials where it has demonstrated similar efficacy to naproxen for pain relief but with an improved gastrointestinal profile (Hawkey et al., 2003; Lohmander et al., 2005). Phase II results with this compound showed an improved gastrointestinal profile compared to naproxen although the primary endpoint of ulcer reduction was not achieved (Lohmander et al., 2005). In the present study, we compared the anti-inflammatory effect of two NOdonating naproxen derivatives, NMI-1182 and AZD3582, with naproxen itself. We also compared the gastric tolerability of the three drugs. Both NMI-1182 and

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AZD3582 are effectively converted in the rat to their parent naproxen as evidenced by the levels of naproxen detected in rat plasma after dosing. The biovavailability of both compounds, however compared to naproxen is slightly less. The observation that NMI-1182 relaxed the rat isolated aorta, whereas AZD3582 did not, suggests that the dinitrate NMI-1182 is converted by the tissue to bioactive NO, while the mononitrate AZD3582 is not in this preparation. One of the main goals of this study was to provide evidence that the NO-donating naproxen derivatives (NMI-1182 and AZD3582) are gastro-protective compared to naproxen. We show that both NMI-1182 and AZD3582 produce significantly less gastric lesions compared to naproxen. The des-NO donor analog of NMI-1182 produced similar gastric damage to naproxen. The only difference in the structure of the des-NO compound from NMI-1182 is a replacement of the NO2 group by H. This provides evidence that the reduction in gastric damage produced by NMI-1182 is due to donation of NO by NMI-1182 and is not due to a pro-drug effect. This is consistent with prior evidence that NO is capable of reducing the gastric damage produced by NSAIDs (Muscara et al., 1998; Fiorucci et al., 2003). As important as it is to demonstrate a reduction in gastric damage produced by NMI-1182 and AZD3582, it is equally important that they retain their ability to act as effective anti-inflammatory agents. As demonstrated, NMI-1182, AZD3582 and naproxen, when given at equimolar doses, had similar activity in the rat paw edema model. In the air pouch model, however, there were some differences noted among the three compounds. Both naproxen and NMI-1182 produced a significant reduction (approximately 50%) in the number of white blood cells infiltrating into the pouch compared to the vehicle control group. AZD3582 on the other hand had no effect on the number of infiltrating cells. One possible reason for this might be found in the PGE2 measurements in the pouch exudates. Naproxen and NMI1182 both produced a 90% or greater reduction in PGE2 levels, whereas AZD3582 produced approximately a 70% reduction in PGE2 levels. It may be necessary therefore to inhibit PGE2 levels almost completely in order to have a significant effect on leukocyte infiltration in this model (Wallace et al., 1999). While not the subject of the present study it is also likely that adding NO-donating ability to NSAIDs will provide cardiovascular benefit in addition to the GI benefit seen with these compounds. This includes anti-thrombotic activity as well as the potential to ameliorate the increase in blood pressure elicited by NSAIDs (Muscara et al., 1998, 2001; Janero et al., 2002; Walford and Loscalzo, 2003; Rossoni et al., 2004). This additional cardiovascular benefit becomes much more pertinent due to the recent concern over cardiovascular safety of both the coxibs and the NSAIDs (Mukherjee et al., 2001; Reicin et al., 2002; Solomon et al., 2004). In conclusion, we have demonstrated that NMI-1182 is an effective anti-inflammatory agent in the rat and compared to its parent, naproxen, it has a much reduced likelihood to produce gastric damage. If this profile can be demonstrated in clinical trials then compounds like NMI-1182 may have considerable therapeutic benefits over traditional NSAIDs or the recently introduced coxibs.

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